US4766205A
Expired / Lapsed
US
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Method for isolation of recombinant polypeptides in biologically active forms
Assignee: Xoma Royalty Corp
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Filing: 1985-11-13
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Granted: 1988-08-23
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Expires: 2005-11-13
Abstract
Abstract Disclosed are improved methods for orienting polypeptide molecules containing multiple disulfide bonds into their native conformation through (1) treating the polypeptide with (a) a denaturing agent and (b) a reducing agent capable of reductively dissociating cystine disulfide bonds; (2) concurrently reducing the concentration of the reducing agent while forming a stable intermediate by reacting a disulfide containing compound with the reduced cysteine moieties of the polypeptide and; (3) forming native cystine disulfide bonds in the presence of a mild oxidizing/reducing environment while dissociating the disulfide-group polypeptide moeities.
Inventors
- 👤 Pradip Ghosh-Dastidar
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 18:51
CPC Classifications
C; C07; C07K; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; C07K14/00; C07K14/415; C07K14/43
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
87 words
A method for establishing a biologically active native conformation in a polypeptide containing multiple disulfide bonds comprising the steps: (1) treating the polypeptide with (a) a denaturing agent and (b) a reducing agent capable of reductively dissociating disulfide bonds; (2) concurrently reducing the concentration of the reducing agent while forming a stable intermediate by reacting a disulfide-group containing compound with the reduced cysteine moieties of the polypeptide; and (3) forming native disulfide bonds in the presence of a mild oxidizing/reducing environment while dissociating the disulfide-group polypeptide moieties.
Claim 2
Independent Claim
87 words
A method for establishing a biologically active native conformation in a polypeptide containing multiple disulfide bonds comprising the steps: (1) treating the polypeptide with (a) a denaturing agent and (b) a reducing agent capable of reductively dissociating disulfide bonds; (2) concurrently reducing the concentration of the reducing agent while forming a stable intermediate by reacting a disulfide-group containing compound with the reduced cysteine moieties of the polypeptide; and (3) forming native disulfide bonds in the presence of a mild oxidizing/reducing environment while dissociating the disulfide-group polypeptide moieties.
Claim 3
Dependent Claim
22 words
The method of claim 1 wherein reducing agent is removed concurrently with formation of a stable intermediate in a gel filtration column.
Claim 4
Dependent Claim
14 words
The method of claim 1 wherein the polypeptide containing multiple disulfide bonds is thaumatin.
Claim 5
Dependent Claim
14 words
The method of claim 2 wherein the polypeptide containing multiple disulfide bonds is thaumatin.
Claim 6
Dependent Claim
11 words
The method of claim 4 wherein the denaturing agent is urea.
Claim 7
Dependent Claim
11 words
The method of claim 4 wherein the reducing agent is β-mercaptoethanol.
Claim 8
Dependent Claim
23 words
The method of claim 4 wherein the disulfide-group containing compound is a member of the group consisting of cystamine, oxidized glutathione and cystine.
Claim 9
Dependent Claim
23 words
The method of claim 4 wherein the mild oxidizing/reducing environment is a solution comprising a weak reducing agent and a weak oxidizing agent.
Claim 10
Dependent Claim
22 words
The method of claim 8 wherein the weak reducing agent is selected from the group consisting of cysteine, reduced glutathione and cysteamine.
Claim 11
Dependent Claim
24 words
The method of claim 8 wherein the weak oxidizing agent is selected from the group consisting of atmospheric oxygen, cystine, oxidized glutathione and cystamine.
Claim 12
Dependent Claim
19 words
The method of claim 4 wherein the mild oxidizing/reducing environment is characterized by a pH of 7 to 9.
Claim 13
Dependent Claim
42 words
The method of claim 4 wherein the denaturing agent is urea, the reducing agent is β-mercaptoethanol, the disulfide-group containing compound is cystamine and the mild oxidizing/reducing environment is a solution comprising cysteine and atmospheric oxygen with a pH of 7 to 9.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-08-23 | 1992-02-22 | 1992-08-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-08-23 | 1996-02-22 | 1996-08-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-08-23 | 2000-02-22 | 2000-08-23 | $7700.00 | Window Closed / Lapsed |